U.S. patent application number 10/099880 was filed with the patent office on 2004-10-07 for anti-amyloid peptide antibody based diagnosis and treatment of a neurological disease or disorder.
This patent application is currently assigned to Cornell Research Foundation, Inc.. Invention is credited to Szabo, Paul, Weksler, Marc E..
Application Number | 20040197831 10/099880 |
Document ID | / |
Family ID | 23057574 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040197831 |
Kind Code |
A1 |
Weksler, Marc E. ; et
al. |
October 7, 2004 |
ANTI-AMYLOID PEPTIDE ANTIBODY BASED DIAGNOSIS AND TREATMENT OF A
NEUROLOGICAL DISEASE OR DISORDER
Abstract
The invention provides a method for assessing risk of a
neurodegenerative disease or disorder in a subject. The method
comprises comparing a level of anti-amyloid peptide antibody in a
biological sample from a subject to a normal level, wherein a lower
level in the biological sample from the subject indicates the
presence of the disease or disorder. In a specific embodiment, the
disease or disorder is Alzheimer's Disease (AD); in a further
specific embodiment, the amyloid peptide is .beta.-amyloid-42
(A.beta..sub.42). In addition, the discovery that certain
neurodegenerative diseases or disorders are associated with a
deficiency of anti-amyloid antibodies provides a method of treating
such a disease or disorder in a subject. This method comprises
administering a therapeutically effective amount of a human
anti-amyloid peptide antibody to a subject believed to suffer from
the immune deficiency or disorder. For example, the disease or
disorder can be Alzheimer's Disease (AD). In such an embodiment,
the amyloid peptide can be .beta.-amyloid-42 (A.beta..sub.42).
Inventors: |
Weksler, Marc E.; (Paris,
FR) ; Szabo, Paul; (Linden, NJ) |
Correspondence
Address: |
DARBY & DARBY P.C.
805 Third Avenue
New York
NY
10022
US
|
Assignee: |
Cornell Research Foundation,
Inc.
|
Family ID: |
23057574 |
Appl. No.: |
10/099880 |
Filed: |
March 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60276659 |
Mar 16, 2001 |
|
|
|
Current U.S.
Class: |
435/7.2 ;
435/7.92 |
Current CPC
Class: |
G01N 33/6896 20130101;
C07K 16/18 20130101; G01N 2800/2821 20130101; A61P 25/28 20180101;
A61K 2039/505 20130101 |
Class at
Publication: |
435/007.2 ;
435/007.92 |
International
Class: |
G01N 033/53; G01N
033/567; G01N 033/537; G01N 033/543 |
Goverment Interests
[0002] This research leading to the present invention was
supported, in part, by National Institutes of Health Grant No. AG
14669. Accordingly, the U. S. Government has certain rights in this
invention.
Claims
1. A method for assessing risk of Alzheimer's Disease in a subject,
which method comprises: determining a level of
anti-.beta.-amyloid-42 (A.beta..sub.42) antibody in a biological
sample selected from the group consisting of blood, serum, and from
a subject, comparing the level of anti-A.beta..sub.42 antibody in
the biological sample from the subject to a normal level determined
from an average of the level of anti-A.beta..sub.42 antibody in a
biological sample from a population consisting of age-matched
normal subjects who do not show any symptoms of neurodegenerative
disease or disorder associated with amyloidosis, wherein a lower
level in the biological sample from the subject indicates the risk
Alzheimer's Disease
2-4. (canceled)
5. The method according to claim 1, which comprises determining the
level of anti-A.beta..sub.42 antibody in the biological sample by
immunoassay.
6. The method according to claim 5, wherein the immunoassay is an
enzyme-linked immunosorbent assay.
7. (canceled)
8. The method according to claim 1, wherein the subject is from a
family that has a member or members with familial Alzheimer's
Disease.
9. The method according to claim 1, wherein the subject is in his
or her seventh or eighth decade of life.
10-15. (canceled)
16. A method for assessing risk of Alzheimer's Disease in a
subject, which method comprises: determining a level of
anti-.beta.-amyloid-42 (A.beta..sub.42) antibody in a biological
sample selected from the group consisting of blood, serum, and
plasma from a subject, wherein the subject does not exhibit
symptoms of cognitive dysfunction or memory dysfunction, comparing
a level of anti-A.beta..sub.42 antibody in a biological sample, to
a normal level determined from an average of the level of
anti-A.beta..sub.42 antibody in a biological sample from a
population consisting of age-matched normal subjects who do not
show any symptoms of associated with Alzheimer's Disease, wherein a
lower level in the biological sample from the subject indicates the
risk of Alzheimer's Disease.
17. The method according to claim 16, wherein the subject is from a
family that has a member or members with familial Alzheimer's
Disease.
18. The method according to claim 16, wherein the subject is in his
or her seventh or eighth decade of life.
19-30. (canceled)
Description
[0001] This application claims priority under 35 U.S.C. .sctn.
1.119(e) to provisional application Serial No. 60/276,659, filed
Mar. 16, 2001, which is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0003] The deposition of amyloid beta 42 amino acid peptide
(A.beta..sub.42), a toxic cleavage product of the transmembrane
amyloid precursor protein (APP), in the brain of patients with
Alzheimer's disease (AD) is thought to play a pathogenic role in
the disease. Much evidence is consistent with this hypothesis: (i)
the appearance of large numbers of amyloid plaques in the brains of
patients with AD, (ii) early onset of AD in persons with trisomy
21, (iii) the early onset of AD in humans with a mutated APP gene,
and (iv) the appearance of amyloid plaques in the brain and
cognitive impairment in mice expressing a mutant, human
APP-transgene. However, at present physicians have no way to
directly detect amyloid plaques, leaving an AD diagnosis solely to
a neurological examination. Except for family history or APOE4
status, physicians cannot predict who is likely to develop AD.
Moreover, until very recently, therapeutic approaches to AD were
limited to maintaining cholinergic function, using
anti-cholinesterase inhibitors such as Aricept, or to attempt to
decrease the generation of AB42 from APP.
[0004] Recently, a new paradigm for the therapy of AD was suggested
by the finding that immunization of APP transgenic mice with
A.beta..sub.42 decreased cerebral amyloid deposits. This suggested
that immune clearance of A.beta..sub.42 from the brain might also
be a rational therapeutic approaches to AD. Subsequently Bard's
group reported that the administration of murine anti-amyloid-beta
peptide (A.beta..sub.42) antibodies to the APP transgenic moused
model of Alzheimer's Disease (AD) decreases cerebral amyloid
deposition.
[0005] While interesting, these results raised the question: if
murine anti-A.beta.42 antibodies can prevent amyloid deposition in
the brains of APP transgenic mice, what is the relationship to
humans? The clinical trial results of treating AD by immunization
with A.beta. peptide dashed hopes for this approach in humans.
Volunteers developed brain inflammation having characteristics of
encephalitis or meningitis, prompting suspension of the trial
(Weiss, The Washington Post, Sunday Mar. 2, 2002, p. A3).
[0006] Accordingly, a significant need remains to diagnose and
treat AD in humans. The present invention addresses this need
through the discovery of naturally occurring anti-A.beta.
antibodies that appear to be protective.
SUMMARY OF THE INVENTION
[0007] There is a need in the art for identifying whether someone
is at risk for, or has, a neurological disease or disorder. For
example, the only definitive diagnosis for Alzheimer's Disease
occurs upon visual inspection of the brain during autopsy; other
than family history or ApoE4 status, there is no way to assess our
individuals of developing AD risk. The invention provides a great
advance: a method for assessing risk of, including diagnosing,
neurological diseases or disorders such as AD, and for effectively
clearing amyloid plaques.
[0008] The present invention advantageously provides method for
assessing risk of a immune deficiency or disorder in a subject,
which leads to a neurodegenerative disease or disorder. This method
comprises comparing a level of anti-amyloid peptide antibody in a
biological sample from a subject to a normal level, wherein a lower
level in the biological sample from the subject indicates the
presence of the disease or disorder. In a specific embodiment, the
disease or disorder is Alzheimer's Disease (AD); in a further
specific embodiment, the amyloid peptide is .beta.-amyloid-42
(A.beta..sub.42).
[0009] The biological sample can be blood, serum, or plasma.
[0010] The normal level can be determined from an average of the
level of anti-amyloid peptide antibody in the biological sample
from a population of age-matched normal subjects who do not show
any symptoms of the immune deficiency or disorder, or from an
average of the level of anti-amyloid peptide antibody in the
biological sample from a population of all subjects, including
subjects who do not show any symptoms of the immune deficiency or
disorder, or from a single normal sample.
[0011] In a preferred aspect, the method comprises determining the
level of anti-amyloid peptide antibody in the biological sample by
immunoassay, e.g., enzyme-linked immunosorbent assay.
[0012] In addition, the discovery of naturally occurring
anti-A.beta. antibodies, which presumably cross the blood brain
barrier with some facility in order to bind to and affect A.beta.,
provides a source of imaging agents, e.g., for position emission
tomography (PET) scanning.
[0013] The surprising observation that certain neurological
diseases or disorders are associated with a deficiency of
anti-amyloid antibodies leads to an advantageous method of treating
such an immune deficiency or disorder in a subject, which leads to
a neurodegenerative disease or disorder. This method comprises
administering a therapeutically effective amount of a human
anti-amyloid peptide antibody to a subject believed to suffer from
the immune deficiency or disorder. For example, the disease or
disorder can be Alzheimer's Disease (AD). In such an embodiment,
the amyloid peptide can be .beta.-amyloid-42 (A.beta..sub.42).
[0014] In a specific embodiment, the antibody is a monoclonal
antibody, e.g., a humanized antibody, or an antibody from and EBV
transformed cell, preferably obtained from a normal patient. In
another embodiment, the antibody is a polyclonal antibody purified
from normal or healthy serum. It is also possible to generate such
an antibody from combinatorial Ig phage display libraries, and from
xeno mice.
[0015] A therapeutically effective amount of the antibody can be an
amount that provides a level of the antibody in a biological sample
from the subject that is at least the same as or greater than a
normal level. The level of therapeutic anti-amyloid peptide
antibody in a biological sample can be determined by immunoassay,
e.g., enzyme-linked immunosorbent assay. Examples of a biological
sample include blood, serum, or plasma, or cerebral spinal fluid
(CSF). In specific embodiments, the normal level is determined from
an average of the level of anti-amyloid peptide antibody in the
biological sample from a population of age-matched normal subjects
or a population of all subjects who do not show any symptoms of the
immune deficiency or disorder.
DETAILED DESCRIPTION
[0016] This invention pertains to the unexpected discovery that
normal humans have anti-amyloid antibodies, and that humans with
low levels of serum anti-A.beta..sub.42 antibodies might be at that
increased risk of AD. The results described in the Examples
demonstrate this. Thus, AD and other neurological diseases or
disorders may be an immune deficiency disease which can be
predicted or diagnosed by detecting low serum levels of
anti-amyloid antibody, and treated by restoring the anti-amyloid
antibody deficit, e.g., to normal levels or above. Preferably, the
"replacement" antibodies are effective in humans, e.g., "human
antibodies".
[0017] A neurological (i.e., neurodegenerative) disease or disorder
is associated with amyloidosis when amyloid deposits or amyloid
plaques are found in or in proximity to tissues affected by the
disease, or when the disease is characterized by overproduction of
a protein, particularly an amyloid protein, that is or can become
insoluble. The amyloid plaques may provoke pathological effects
directly or indirectly by known or unknown mechanisms. Examples of
amyloid diseases include, but are not limited to, systemic
diseases, such as chronic inflammatory illnesses, multiple myeloma,
macroglobulinernia, familial amyloid polyneuropathy (Portuguese)
and cardiomyopathy (Danish), systemic senile amyloidosis, familial
amyloid polynephropathy (Iowa), familial amyloidosis (Finnish),
Gerstmann-Straussler-Scheinker syndrome, familial amyloid
nephropathy with urticaria and deafness (Muckle-Wells syndrome),
medullary carcinoma of thyroid, isolated atrial amyloid, and
hemodialysis-associated amyloidosis (HAA); and amyloid associated
neurodegenerative diseases.
[0018] As noted above, in addition to systemic amyloidosis, the
present invention relates particularly to neurodegenerative
diseases involving amyloidosis. The term "neurodegenerative
disease" (or "neurological disease") refers to a disease or
disorder of the nervous system, particularly involving the brain,
that manifests with symptoms characteristic of brain or nerve
dysfunction, e.g., short-term or long-term memory lapse or defects,
dementia, cognition defects, balance and coordination problems, and
emotional and behavioral deficiencies. Such diseases are
"associated with amyloidosis" when histopathological (biopsy)
samples of brain tissue from subjects who demonstrate such symptoms
would reveal amyloid plaque formation. As biopsy samples from
brain, especially human brain, are obtained with great difficulty
from living subjects or might not be available at all, often the
association of a symptom or symptoms of neurodegenerative disease
with amyloidosis is based on criteria other than the presence of
amyloid deposits, such as plaques or fibrils, in a biopsy sample.
Thus, particularly with respect to AD, traditional diagnosis
depends on symptomology and, if relevant, family history. In
clinical practice a physician will diagnose Alzheimer's Disease on
the basis of symptoms of senile dementia, including cognitive
dysfunction, retrograde amnesia (loss of memory for recent events),
progressive impairment of remote memory, and possibly depression or
other neurotic syndromes. The individual presents with slow
disintegration of personality and intellect. Imaging may reveal
large cell loss from the cerebral cortex and other brain areas. AD
differs from senile dementia, however, by age of onset: AD is
likely to occur in the fifth or sixth decade, whereas senile
dementia occurs in the eighth decade or later.
[0019] This invention provides a method for diagnosing AD patients
or identifying those at risk of developing AD. In particular, low
serum levels of anti-amyloid, e.g., A.beta..sub.42, antibodies
indicates a predisposition to developing or the presence of a
neurodegenerative disease or disorder. The key advantage over
current clinical diagnostic practice is early detection: detecting
low levels of anti-A.beta. antibodies can predict the onset of AD
before the appearance of symptoms such as cognitive and memory
dysfunction. Thus, low serum anti-AD antibody levels are a risk
factor for AD.
[0020] Furthermore, because the anti-A.beta. antibodies presumably
bind amyloid in the brain, they must be able to cross the blood
brain barrier and recognize the A.beta. with a high degree of
specificity (to avoid undesirable cross-reaction with healthy brain
tissues, which could cause inflammation). Such targeting specifity
renders the natural antibodies of the invention particularly useful
for diagnostic imaging of amyloid plaques in human brain. By
labeling the antibodies with an appropriate radioisotope, it is
possible to use imaging techniques like PET for a definitive
diagnosis of a neurodegenerative disease, such as AD.
[0021] In a specific embodiment, according to the present invention
the neurodegenerative disease associated with amyloidosis is
Alzheimer's disease (AD), a condition that includes sporadic AD,
ApoE4-related AD, other mutant APP forms of AD (e.g., mutations at
APP717, which are the most common APP mutations), mutant PS1 forms
of familial AD (FAD) (see, WO 96/34099), mutant PS2 forms of FAD
(see, WO 97/27296), and alpha-2-macroglobulin-polymorphism-related
AD. In other embodiments, the disease may be the rare Swedish
disease characterized by a double KM to NL mutation in amyloid
precursor protein (APP) near the amino-terminus of the .beta.AP
portion of APP (Levy et al., 1990, Science 248:1124-26). Another
such disease is hereditary cerebral hemorrhage with amyloidosis
(HCHA or HCHWA)-Dutch type (Rozemuller et al., 1993, Am. J. Pathol.
142:1449-57; Roos et al., 1991, Ann. N.Y. Acad. Sci. 640:155-60;
Timmers et al., 1990, Neurosci. Lett. 118:223-6; Haan et al., 1990,
Arch. Neurol. 47:965-7). Other such diseases known in the art and
within the scope of the present invention include, but are not
limited to, sporadic cerebral amyloid angiopathy, hereditary
cerebral amyloid angiopathy, Downs syndrome, Parkinson-dementia of
Guam, and age-related asymptomatic amyloid angiopathy (see, e.g.,
Haan and Roos, 1990, Clin. Neurol. Neurosurg. 92:305-310; Glenner
and Murphy, 1989, N. Neurol. Sci. 94:1-28; Frangione, 1989, Ann.
Med. 21:69-72; Haan et al., 1992, Clin. Neuro. Neurosurg. 94:317-8;
Fraser et al., 1992, Biochem. 31:10716-23; Coria et al., 1988, Lab.
Invest. 58:454-8). The actual amino acid composition and size of
the .beta.AP involved in each of these diseases may vary, as is
known in the art (see above, and Wisniewski et al., 1991, Biochem.
Biophys. Res. Commun. 179:1247-54 and 1991, Biochem. Biophys. Res.
Commun. 180:1528 [published erratum]; Prelli et al., 1990, Biochem.
Biophys. Res. Commun. 170:301-307; Levy et al., 1990, Science
248:1124-26).
Amyloid
[0022] The terms "amyloid," "amyloid plaque," and "amyloid fibril"
refer generally to insoluble proteinaceous substances with
particular physical characteristics independent of the composition
of proteins or other molecules that are found in the substance.
Amyloid can be identified by its amorphous structure, eosinophilic
staining, changes in thioflavin fluorescence, and homogeneous
appearance. Protein or peptide components of amyloid are termed
herein "amyloid polypeptides," and include, but are not limited to,
.beta.-amyloid peptide (A.beta.), including synthetic .beta.APs
corresponding to the first 28, 40, or 42 amino acids of A.beta.,
i.e., A.beta.(1-28) or A.beta.28, A.beta.(1-40) or A.beta.40,
A.beta.(1-42) or A.beta.42, respectively, as well as a synthetic
.beta.AP corresponding to amino acids 25-35 of A.beta., i.e.,
A.beta..sub.25-35. Other amyloid peptides include scrapie protein
precursor or prion protein (associated with Creuzfeldt-Jacob's
disease); synuclein (associated with Parkinson's disease),
Huntington's protein (associated with Huntington's chorea),
immunoglobulin, including .kappa. or .lambda. light or heavy
chains, or fragments thereof, produced by myelomas; serum amyloid
A; .beta.2-microglobulin; ApoA1; gelsolin; cystatin C;
(pro)calcitonin; atrial natururetic factor; islet amyloid
polypeptide, also known as amylin (see, Westermark et al., Proc.
Natl. Acad. Sci. USA 84:3881-85, 1987; Westermark et al., Am. J.
Physiol. 127:414-417, 1987; Cooper et al., Proc. Natl. Acad. Sci.
USA 84:8628-32, 1987; Cooper et al., Proc. Natl. Acad. Sci. USA
85:7763-66, 1988; Amiel, Lancet 341:1249-50, 1993); and the like.
In a specific aspect, the term "amyloid" is used herein to refer to
substances that contain A.beta.. "Amyloidosis" refers to the in
vivo deposition or aggregation of proteins to form amyloid plaques
or fibrils.
[0023] The 42 amino acid (4.2 kDa) beta-Amyloid Peptide
A.beta..sub.42 or .beta.AP) derives from a family of larger Amyloid
Peptide Precursor (APP) proteins (Glenner and Wong, 1984, Biochem.
Biophys. Res. Commun. 120:885-890; Glenner and Wong, 1984, Biochem.
Biophys. Res. Commun. 122:1131-35; Goldgaber et al., 1987, Science
235:8778-8780; Kang et al., 1987, Nature 325:733-736; Robakis et
al., 1987, Proc. Natl. Acad. Sci. USA 84:4190-4194; Tanzi et al.,
1987, Science 235:880-884). APP 25 is a transmembrane protein found
in a number of isoforms, which in general are referred to herein as
full length APP (flAPP). In addition, there is a soluble form of
APP (sAPP.alpha.), formed by the action of .alpha.-secretase
(discussed supra).
[0024] The "level of A.beta." in a biological sample can be
detected by any method known in the art, including by not limited
to immunoassay (as exemplified infra), biochemical analysis (e.g.,
purification, gel electrophoresis, quantitative amino acid sequence
analysis or composition analysis, Congo red or Thioflavin-T
staining, and the like), or other methods known to detect A.beta..
In particular, fluorescence methods using Thioflavin T are used to
detect aggregated peptide. A "biological sample" includes, but is
not limited to body fluids (blood, blood cells, plasma, serum,
cerebrospinal fluid, urine), tissues (e.g., spinal chord, nerves,
etc.), or organs (preferably brain, but also including liver,
kidney, pancreas, etc.).
Antibodies to Amyloid Peptides
[0025] According to the invention, anti-amyloid peptide, i.e.,
produced recombinantly or by chemical synthesis, and fragments or
other derivatives or analogs thereof, including fusion proteins,
may be used as an antigen to detect and purify, and in some
instances as an immunogen to generate, natural antibodies that
recognize the amyloid peptide. As used herein, the term "natural
antibody" (whether singular or plural) refers to an antibody having
the functional characteristics of serum antibodies found in normal
human subjects. Such antibodies can include but are not limited to
polyclonal, monoclonal, chimeric, single chain, Fab fragments, and
an Fab expression library. The anti-amyloid peptide antibodies of
the invention may be cross reactive, e.g., they may recognize
amyloid peptide from different species. Polyclonal antibodies have
greater likelihood of cross reactivity. Alternatively, an antibody
of the invention may be specific for a single form of amyloid
peptide, such as an anti-human .beta.amyloid-42 peptide. Such an
antibody is effective in human, e.g., a human (including humanized
and chimeric) antibody.
[0026] Various procedures known in the art may be used for the
production of polyclonal antibodies to amyloid peptide. Sera from a
normal individual or pooled sera from a group of normal
individuals, provides a source of amyloid protective anti-amyloid
antibody. Purification of antibodies can be achieved by standard
techniques for immunoglobulin production, as is well known in the
art.
[0027] For preparation of monoclonal antibodies directed toward the
amyloid peptide, any technique that provides for the production of
antibody molecules by continuous human or human hybridoma cell
lines in culture may be used. These include, but are not limited,
to the hybridoma technique originally developed by Kohler and
Milstein (Nature 256:495-497, 1975), as well as the trioma
technique, the human B-cell hybridoma technique (Kozbor et al.,
Immunology Today 4:72, 1983; Cote et al., Proc. Natl. Acad. Sci.
U.S.A. 80:2026-2030, 1983), and the EBV-hybridoma technique to
produce human monoclonal antibodies (Cole et al., in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96,
1985). In an additional embodiment of the invention, monoclonal
antibodies can be produced in germ-free animals (PCT Publication
No. WO 89/12690). In fact, according to the invention, techniques
developed for the production of "chimeric antibodies" (Morrison et
al., J. Bacteriol. 1 59:870, 1 984; Neuberger et al., Nature
312:604-608, 1984; Takeda et al., Nature 314:452-454, 1985) by
splicing the genes from a mouse antibody molecule specific for an
anti-amyloid peptide polypeptide together with genes from a human
antibody molecule of appropriate biological activity can be used;
such antibodies are within the scope of this invention. Such human
or humanized chimeric antibodies are preferred for use in therapy
of human diseases or disorders (described infra), since the human
or humanized antibodies are much less likely than xenogenic
antibodies to induce an immune response, in particular an allergic
response, themselves.
[0028] In a specific embodiment, anti-amyloid peptide antibody can
be obtained from EBV transformed cells isolated from individuals
who are protected from amyloidosis, i.e., have normal or elevated
anti-amyloid antibody levels. Alternatively the antibodies can be
isolated from normal serum using any of the multitude of techniques
known in the art. These "natural" antibodies have the advantage of
expected efficacy: they are protective in normal subjects, and
their absence can lead to development of degenerative neuropathies
characterized by amyloidosis.
[0029] According to the invention, techniques described for the
production of single chain antibodies (U.S. Pat. Nos. 5,476,786 and
5,132,405 to Huston; U.S. Pat. No. 4,946,778) can be adapted to
produce amyloid peptide specific single chain antibodies. Indeed,
these genes can be delivered for expression in vivo. An additional
embodiment of the invention utilizes the techniques described for
the construction of Fab expression libraries (Huse et al., Science
246:1275-1281, 1989) to allow rapid and easy identification of
monoclonal Fab fragments with the desired specificity for an
amyloid peptide, or its derivatives, or analogs.
[0030] Antibody fragments which contain the idiotype of the
antibody molecule can be generated by known techniques. For
example, such fragments include but are not limited to: the
F(ab').sub.2 fragment which can be produced by pepsin digestion of
the antibody molecule; the Fab' fragments which can be generated by
reducing the disulfide bridges of the F(ab').sub.2 fragment, and
the Fab fragments which can be generated by treating the antibody
molecule with papain and a reducing agent.
[0031] Assays for the anti-.beta. amyloid antibody can be
accomplished by techniques known in the art, e.g.,
radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitin reactions, immunodiffusion assays, in situ immunoassays
(using colloidal gold, enzyme or radioisotope labels, for example),
Western blots, precipitation reactions, agglutination assays (e.g.,
gel agglutination assays, hemagglutination assays), complement
fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody
binding is detected by detecting a label on the primary antibody.
In another embodiment, the primary antibody is detected by
detecting binding of a secondary antibody or reagent to the primary
antibody. In a further embodiment, the secondary antibody is
labeled. Many means are known in the art for detecting binding in
an immunoassay and are within the scope of the present invention.
For example, to select antibodies which recognize a specific
epitope of an amyloid peptide, one may assay generated hybridomas
for a product which binds to an amyloid peptide fragment containing
such epitope.
Pharmaceutical Compositions and Administration
[0032] As noted above, clinical testing of an
A.beta..sub.42-peptide vaccine resulted in brain inflammation in a
number of patients. The present invention involves recognition that
individuals with normal levels of anti-amyloid antibodies seem to
be protected from neurodegenerative disease without developing
brain inflammation. Thus, delivery of natural anti-amyloid
antibodies, i.e., passive immunization, to subjects at risk for or
suffering from a neurodegenerative disease, e.g., AD, has greater
potential for safety as well as efficacy.
[0033] The anti-amyloid peptide antibody of the invention can be
formulated in a pharmaceutical composition with a pharmaceutically
acceptable carrier. The phrase "pharmaceutically acceptable" refers
to molecular entities and compositions that are physiologically
tolerable and do not typically produce an allergic or similar
untoward reaction, such as gastric upset, dizziness and the like,
when administered to a human. Preferably, as used herein, the term
"pharmaceutically acceptable" means approved by a regulatory agency
of the Federal or a state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in
animals, and more particularly in humans. The term "carrier" refers
to a diluent, adjuvant, excipient, or vehicle with which the
compound is administered. Such pharmaceutical carriers can be
sterile liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, sesame oil and the like. Water or
aqueous solution saline solutions and aqueous dextrose and glycerol
solutions are preferably employed as carriers, particularly for
injectable solutions. Suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W.
Martin.
[0034] A composition comprising "A" (where "A" is a single protein,
DNA molecule, vector, recombinant host cell, etc.) is substantially
free of "B" (where "B" comprises one or more contaminating
proteins, DNA molecules, vectors, etc.) when at least about 75% by
weight of the proteins, DNA, vectors (depending on the category of
species to which A and B belong) in the composition is "A".
Preferably, "A" comprises at least about 90% by weight of the A+B
species in the composition, most preferably at least about 99% by
weight. It is also preferred that a composition, which is
substantially free of contamination, contain only a single
molecular weight species having the activity or characteristic of
the species of interest.
[0035] According to the invention, the anti-amyloid antibody
formulated in a pharmaceutical composition of the invention can be
introduced parenterally, transmucosally, e.g., orally (per os),
nasally, or rectally, or transdermally. Parental routes include
intravenous, intra-arteriole, intramuscular, intradermal,
subcutaneous, intraperitoneal, intraventricular, and intracranial
administration. Preferably, administration is directly into the
cerebrospinal fluid, e.g., by a spinal tap.
[0036] In another embodiment, the therapeutic compound can be
delivered in a vesicle, in particular a liposome (see Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss: New York, pp. 353-365 (1989); Lopez-Berestein,
ibid., pp. 317-327; see generally ibid.). To reduce its systemic
side effects, this may be a preferred method for introducing the
compound.
[0037] In yet another embodiment, the therapeutic compound can be
delivered in a controlled release system. For example, a
polypeptide may be administered using intravenous infusion with a
continuous pump, in a polymer matrix such as poly-lactic/glutamic
acid (PLGA), a pellet containing a mixture of cholesterol and the
anti-amyloid peptide antibodycompound (SilasticR.TM.; Dow Corning,
Midland, Mich.; see U.S. Pat. No. 5,554,601) implanted
subcutaneously, an implantable osmotic pump, a transdermal patch,
liposomes, or other modes of administration. In one embodiment, a
pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed.
Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek
et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,
polymeric materials can be used (see Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton,
Fla. (1 974); Controlled Drug Bioavailability, Drug Product Design
and Performance, Smolen and Ball (eds.), Wiley: N.Y. (1 984);
Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61
(1983); see also Levy et al., Science 228:190 (1985); During et
al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg.
71:105 (1989)). In yet another embodiment, a controlled release
system can be placed in proximity of the therapeutic target, i.e.,
the brain, thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release,
supra, vol. 2, pp.115-138 (1 984)). Preferably, a controlled
release device is introduced into a subject in proximity of the
site of amyloidosis. Other controlled release systems are discussed
in the review by Langer (Science 249:1527-1533 (1990)).
[0038] The therapeutic compositions and regimens of the invention
are useful for treating a neurological disease or disorder
associated with a deficiency of anti-amyloid antibodies. Thus, the
"neurological diseases or disorder" of the invention is a
neuropathy involving amyloid deposition, and associated with
specific or general immunodeficiency. These diseases include, but
are not limited to, Alzheimer's Disease; Kuru, Creuzdfelt-Jacob's
disease, and other spongiform encephalopathies; Parkinson's
Disease; and Huntington's chorea. The therapeutic regimens,
specifically passive immunization with protective human antibodies
against the amyloid peptide, are unexpectedly effective because
they preferably employ anti-amyloid antibodies from normal
individuals who seem to have protective immunity against amyloid
deposition. These antibodies supplement the deficiency associated
with the neurological disease or disorder.
Dosage and Regimen
[0039] A constant supply of the anti-amyloid peptide antibody can
be ensured by providing a therapeutically effective dose (i.e., a
dose effective to induce metabolic changes in a subject) at the
necessary intervals, e.g., daily, every 12 hours, etc. These
parameters will depend on the severity of the disease condition
being treated, other actions, such as diet modification that are
implemented, the weight, age, and sex of the subject, and other
criteria, which can be readily determined according to standard
good medical practice by those of skill in the art. Preferably, the
anti-amyloid peptide antibody is administered for at least ten
days, more preferably at least 100 days, and more preferably still,
for the life of the recipient.
[0040] The term "prevent the onset of" means to prophylactically
interfere with a pathological mechanism that results in the disease
or disorder. In the context of the present invention, such a
pathological mechanism can be an increase in processing of the
amyloidogenic form of APP; dysregulation of A.beta. clearance; or
some combination of the two. The term "ameliorate" means to cause
an improvement in a condition associated with the disease or
disorder. In the context of the present invention, amelioration
includes a reduction in the level of A.beta., regulation of the
formation of A.beta., decrease in aggregation of A.beta. or the
formation of amyloid plaques, or improvement of a cognitive defect
in a subject suffering from a disease or disorder associated with
amyloidosis, e.g., Alzheimer's disease or an animal model of
Alzheimer's disease. The phrase "therapeutically effective amount"
or "dose" is used herein to mean an amount or dose sufficient to
reduce the level of amyloid peptide, e.g., by about 10 percent,
preferably by about 50 percent, and more preferably by about 90
percent. Preferably, a therapeutically effective amount can
ameliorate or prevent a clinically significant deficit in the
activity, function, and response of the host. Alternatively, a
therapeutically effective amount is sufficient to cause an
improvement in a clinically significant condition in the host.
[0041] A subject who "has an increased risk of developing" a
neurological disease or disorder associated with amyloidosis may
have a genetic predisposition to developing an amyloidosis, such as
a person from a family that has members with familial Alzheimer's
Disease (FAD). Alternatively, someone in his or her seventh or
eighth decade is at greater risk for age-related AD.
[0042] A subject who "shows a symptom of" a neurological disease or
disorder associated with amyloidosis presents with a symptom or
complaint found in subjects who have or have had such a disease or
disorder. For example, in Alzheimer's Disease, these symptoms can
include development of dementia, memory defects, and the like in
the fifth and sixth decade, as discussed above.
[0043] An "A.beta. level reducing dose" is an amount of
anti-amyloid peptide antibody that causes a decrease in the level
of A.beta.. Dosages can range from about 0.5 .mu.g anti-amyloid
peptide antibody per kg body weight to (.mu.g/kg) to about 50
mg/kg, per day; preferably from about 5 .mu.g/kg to about 10 mg/kg,
per day. The amount of anti-amyloid peptide antibody used to
decrease the level of A.beta. can be an amount corresponding to the
level of anti-amyloid peptide antibody in a biological sample,
especially blood (including plasma and serum) and CSF, from a
normal subject.
[0044] A subject in whom administration of the anti-amyloid peptide
antibody is an effective therapeutic regiment for a disease or
disorder associated with amyloidosis is preferably a human.
[0045] "Reducing a level of amyloid-.beta. (A.beta.) peptides"
specifically refers to decreasing the amount of A.beta.40 or,
preferably, A.beta.42, or more preferably, both, in vivo.
A.beta.can accumulate in blood, cerebrospinal fluid, or organs. The
primary organ of interest for reducing the level of A.beta. is
brain, but A.beta. levels may also be reduced in body fluids,
tissues, and/or other organs by the practice of this invention.
[0046] As used herein, the term "about" or "approximately" means
within 50% of a given value, preferably within 20%, more preferably
within 10%, more preferably still within 5%, and most preferably
within 1% of a given value. Alternatively, the term "about" or
"approximately" means that a value can fall within a scientifically
acceptable error range for that type of value, which will depend on
how quantitative a measurement can be given the available
tools.
EXAMPLE 1
Low Anti-Amyloid Antibodies in Alzheimer's Disease
[0047] Late onset Alzheimer's disease (AD) is associated with
increased deposits of .beta.-amyloid peptide (A.beta..sub.42) in
the brain although serum A.beta..sub.42 levels remain normal.
Transgenic mice expressing a mutated, human amyloid precursor
protein (APP) gene develop deposits of A.beta. in their brain as
they age and are a useful experimental model of human AD. When such
transgenic mice are immunized with A.beta..sub.42 in a modified
Freund's adjuvant or passively immunized by giving murine
anti-A.beta..sub.42, deposition of A.beta..sub.42 in the brain is
significantly reduced (Schenk et al, Nature 1999, 400:173-7; Bard
et al, Nat. Med. 2000, 6:916-9). Recently, immune-mediated
reduction of cerebral AP was shown to limit cognitive loss in
treated compared to control transgenic mice expressing a mutated,
human, APP gene (Morgan et al, Nature 2000, 408:982-5; Janus et al,
Nature 2000, 408:979-82). These results have led to clinical trials
testing whether active immunization with A.beta..sub.42 can convey
therapeutic benefits to AD patients. The potential success of this
strategy is thought to depend on the capacity of the immune system
in AD patients to produce anti-A.beta..sub.42.
[0048] The ability of anti-A.beta..sub.42 antibodies to decrease
the deposition of the cerebral amyloid peptides and thereby prevent
or delay the development of AD-like neuropathology in mice raised,
but left unanswered, the question of whether a similar mechanism
could operate in AD. In particular, we considered whether AD
patients might have lower levels of spontaneous serum
anti-AB.sub.42 antibodies than comparably aged normal controls. We
measured serum levels of anti-A.beta..sub.42 antibodies in
fresh-frozen serum from 39 patients with late-onset AD (mean
age=73.2.+-.8.2) and 39 elderly adults without AD (mean
age=77.7.times.10.8). Patients with AD were living in the
community, had mild to moderate intellectual impairment, and met
the NINCDS-ADRDA criteria for Probable AD. Serum from normal
controls was obtained from elderly volunteers and spouses of
patients who gave informed consent to undergo neuropsychological
testing and blood drawing. The serum levels of anti-A.beta..sub.42
antibodies were measured by an ELISA using a commercially available
A.beta..sub.42 (Biosource International, Camarillo, Calif., USA).
Anti-A.beta..sub.42 antibody levels in serum from study subjects
were determined in three independent assays. In each assay, the
antibody level in the subject was compared to that of the same
reference serum from an individual without AD. Because
anti-A.beta..sub.42 antibodies levels in serum varied 1000-fold and
were not normally distributed, a statistically stringent analysis
of anti-A.beta..sub.42 antibody concentrations above or below the
reference serum was carried out using the non-parametric Chi square
as well as a rank order Mann-Whitney test or T-tests on log
transformations of the data. Seventy-seven percent of AD patients
in our sample had low levels of serum anti-A.beta..sub.42 antibody,
below that of the standard serum, a significantly smaller
percentage than that found in healthy, elderly controls. The mean
reciprocal titers of both anti-A.beta. total Ig and anti-A.beta.
IgG were significantly lower in AD patients than elderly adults
without AD as determined by T-test and Mann-Whitney test (Table 1).
The difference was most significant in the case of anti-A.beta. IgG
antibodies.
1TABLE 1* Ig and IgG anti-A.beta..sub.42 Antibody Titer is Lower in
Serum from AD Patients than Elderly Controls* (Reciprocal Titer and
95% confidence interval) Statistical Test Elderly Controls AD
Patients Mann- (n = 39) (n = 39) T Test Whitney Ig anti- 117
(70-200) 50 (30-80) p < 0.02 p < 0.04 A.beta..sub.42 IgG
anti- 724 (440-1200) 295 (180-490) p < 0.01 p < 0.02
A.beta..sub.42 *Coded sera were tested for anti-A.beta..sub.42
peptide antibodies using an ELISA assay. Microtiter wells were
coated with A.beta..sub.42 washed, blocked, and incubated with
3-fold dilutions of the serum. The plates were then washed and
incubated with alkaline phosphatase-conjugated goat anti-human
immunoglobulin antibody. The plates were washed and incubated with
enzyme substrate p-Nitrophenylphosphate. #The concentration of
anti-A.beta..sub.42 peptide antibodies was the dilution of serum
that gave half maximal binding and classified as being more or less
than concentration of anti-A.beta..sub.42 peptide antibodies in the
reference serum.
[0049] The apolipoprotein E (ApoE)-e4 allele, a recognized risk
factor for AD, was present in 51% of the patients with AD and 15%
of the normal elderly in this study. The percentage of individuals
with low serum anti-A.beta..sub.42 antibody levels was greater in
ApoE-e4 carriers. However, in non-ApoE-e4 carriers, a low level of
serum anti-A.beta..sub.42 IgG antibodies was significantly
associated with AD (Table 2). Thus, lower serum anti-A.beta..sub.42
antibodies is significantly associated with AD independent of ApoE
genotype.
2TABLE 2 IgG anti-A.beta..sub.42 Antibody Titer is Lower in Serum
from in AD Patients than in Elderly Controls not Expressing
Apoprotein E4 (Reciprocal Titer and 95% confidence interval)
Controls E4-Negative AD E4-Negative Statistic (n = 33) (n = 19) T
Test Mann-Whitney Test Ig Anti-A.beta..sub.42 112 (79-190) 67
(34-132) p < 0.230 p < 0.240 IgG anti-A.beta..sub.42 745
(447-1251) 297 (152-580) p < 0.025 p < 0.033
*Anti-A.beta..sub.42 antibodies in patients and age-matched
controls expressing apolipoprotein E4 gene were compared. The level
of serum anti-A.beta..sub.42 antibodies was determined and analyzed
as described in the Legend to Table 1. In addition, the values
above and below the median value of the total sample were
counted.
[0050] The association of a low level of serum anti-A.beta..sub.42
antibodies with AD raises the interesting possibility that AD, and
conceivably other forms of dementia, may be associated with an
impaired immune response to A.beta..sub.42. Previously published
results are consistent with this hypothesis (Trieb et al,
Neurobiol. Aging 1996, 17:541-7). Thus, T cells from AD patients
had no proliferative response or a very low proliferative response
when cultured with A.beta..sub.42. In contrast, T cells from young
or old healthy individuals, cultured similarly, had a significant
proliferative response. This impaired proliferative response of T
cells from AD patients was not due to a generalized defect as T
cells from AD patients responded as well as those from young and
old individuals when cultured with the non-specific mitogen, OKT-3
antibody.
[0051] Our present data have implications for clinical trials now
underway using active immunization with A.beta..sub.42. Thus, AD
patients with low serum levels of spontaneous anti-A.beta..sub.42
antibodies may not, despite immunization with A.beta..sub.42,
develop therapeutic titers of anti-A.beta..sub.42 antibodies. This
may reflect either impaired immunity to or immune "tolerance" of
A.beta..sub.42. Impaired immunity due to decreased T helper
function is suggested by the decreased proliferative response of T
cells from AD patients to A.beta..sub.42. For this reason, it may
be difficult to induce an anti-A.beta..sub.42 antibody response in
AD patients. However, passive immunity, induced by the
administration of human anti-A.beta..sub.42 antibodies, especially
antibodies isolated from healthy humans or healthy humans' B cells,
would not depend on the recipient's immune response to
A.beta..sub.42. Thus, passive immunization could represent a viable
approach to the treatment or prevention of AD in those patients who
cannot generate a significant anti-A.beta..sub.42-anti- body
response.
EXAMPLE 2
Evidence of Lower Anti-Amyloid Antibody Levels in Other
Neurological Diseases
[0052] Forty one patients with AD had significantly (p<0.01)
lower serum anti-A.beta..sub.42 antibody levels than did 42
age-matched healthy controls or one arbitrarily selected standard
(Table 3). Patients with other neurological diseases were also
compared to age-matched healthy controls. Seven of eight patients
with other neurological diseases had lower serum
anti-A.beta..sub.42 antibody levels than did the 21 controls in
this experiment. While not statistically significant, these data
indicate that significantly lower serum anti-A.beta..sub.42
antibody levels in AD are specific for AD or for neurological
disease.
3TABLE 3 1.sup.st Set AD NORMAL Other Cell < 5 Fisher Above MW 2
10 0 Exact Below MW 7 2 0 2 tailed P value: 0.009 2.sup.nd Set AD
NORMAL Other Chi Square with Above MW 6 9 1 Yates Correction: 0.73
for Ad VS Normal Below MW 17 12 7 Cell < 5 Fisher Exact 2 tailed
P value: 0.2 for Other Vs Normal 3.sup.rd Set AD NORMAL Other Cell
< 5 Fisher Above MW 2 4 0 Exact Below MW 7 5 0 2 tailed P value:
0.62 4.sup.th Set AD NORMAL Chi Square with Above MW 10 23 Yates
Correction: Below MW 31 19 0.009 AD = test results for samples from
Alzheimer Disease patients Normal = test results for samples from
normal patients Other = test results from patients with other
neuropathies (Parkinson's; Huntington's) MW = arbitrary normal
serum sample.
EXAMPLE 3
Reproducibility of ELISA Results
[0053] To find out the reproducibility of the ELISA essay,
triplicates of sera from 3 normal subjects were coded and assayed
blindly. We were able to group the 9 specimens to identify
specimens coming from each of the 3 subjects. Furthermore, when the
assay was performed the following day, the triplicate from the same
subjects could again be identified. The rank of the subjects was
identical on day 1 and day 2 (Table 4).
[0054] Day 1;
4 Sample Titer Standard Deviation 43 0.26 0.03 0.28 0.32 61 1.06
0.10 1.20 1.25 1 11.20 1.59 13.75 14.12
[0055] Day 2:
5 Sample Titer Standard Deviation 43 0.20 0.17 0.20 0.50 61 1.20
0.57 1.24 2.20 1 16.70 1.31 18.50 19.26
[0056] These data show that the ELISA essay is highly reproducible.
It is not certain whether the distribution of results on the same
serum changes in repeated essay. Thus, the standard deviation in 2
out of 3 samples increased on day 2, while one sample remained
approximately the same.
[0057] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0058] It is further to be understood that all values are
approximate, and are provided for description.
[0059] Patents, patent applications, publications, product
descriptions, and protocols are cited throughout this application,
the disclosures of which are incorporated herein by reference in
their entireties for all purposes.
* * * * *